The identification and quantification of proteins in complex biological samples is critically important to biological and biomedical research. Tandem mass spectrometry (MS/MS) is a technique capable of identifying large numbers of proteins in complex biological samples. In a typical experiment, proteins are digested with site-specific reagents to produce smaller peptides, the peptides are separated via high-performance liquid chromatography (HPLC) and the separated peptides are injected onto a tandem mass spectrometer. In such an experiment, as the peptides elute from the liquid chromatography (LC) column, they are subjected to an ionizing voltage and are introduced into the near vacuum environment of the tandem mass spectrometer. A survey scan (i.e., a first mass spectrum or MS1) is obtained to determine the mass-to-charge ratio (m/z) of the intact peptides that entered the tandem mass spectrometer. The ionized peptides detected in the first mass spectrum may be referred to as precursor ions. One or more of the precursor ions in the first mass spectrum is selected, sequentially isolated, fragmented and the resulting fragment ion m/z values determined in a second mass spectrum (i.e., MS/MS or MS2). Generally, only the most intense precursor ions are selected for generation of the second mass spectrum. The fragment ions detected in the second mass spectrum may be referred to as product ions. This process is repeated to automatically acquire MS/MS spectra of as many of the precursor ions as possible. The charge state and peptide mass are obtained from the first mass spectrum while the fragmentation pattern is recorded in the second mass spectrum. With this information it is possible to identify the peptide and protein of origin, e.g., by comparison to peptide fragmentation patterns stored in a database. The procedure is repeated time and time again, as quickly as possible, to sample as many peptides in the sample as possible.
As described above, tandem mass spectrometry permits the identification of peptides and proteins. However, it is also important to quantify peptides and proteins, e.g., to compare the amount of a particular protein among two or more different samples (e.g., replicate samples, samples subject to different experimental conditions, samples from different time points, samples from different laboratory animals, etc.). One approach for quantitative proteomics involves combining tandem mass spectrometry with stable isotope labeling techniques. Depending upon the type of stable isotope labeling technique, quantification can be obtained from MS1 spectra or MS2 spectra or both.
As an example of a stable isotope labeling technique that can provide quantification from MS1 spectra, one sample (e.g., a sample containing peptides) is left with its natural isotope abundance (unlabeled) and the other sample is made to incorporate a heavy isotope (labeled). The two samples are mixed and analyzed simultaneously (i.e., by injection into the HPLC and tandem mass spectrometer). A labeled peptide and its unlabeled counterpart have the same chemical formula and same chemical structure and thus, will elute together from the LC column. However, the introduction of the isotope label results in a predictable mass difference between such peptides, resulting in the appearance of “peptide pairs” in the MS1 spectra. By comparing the signal intensities of the labeled and unlabeled peptide pairs, relative quantification is obtained. Absolute quantification may be obtained if the labeled sample is replaced with synthetic peptides of known quantity. In either case, the quantitative values are obtained from the MS1 spectra. Techniques for stable isotope labeling and quantification from MS1 spectra include, for example, stable isotope labeling with amino acids in cell culture (SILAC). In a “two-plex” SILAC experiment, two different samples are compared simultaneously by using light (e.g., unlabeled) and heavy labeled peptides. In a “three-plex” SILAC experiment, three different samples are compared simultaneously by using light (e.g., unlabeled), medium and heavy labeled peptides.
As an example of a stable isotope labeling technique that can provide quantification from MS2 spectra, samples (e.g., samples containing peptides) are labeled with a set of isobaric tags. The samples, each labeled with a different isobaric tag from the set, are mixed and analyzed simultaneously. The isobaric tags in the set have the same chemical formula and same chemical structure and thus, the different versions of a labeled peptide (e.g., a peptide labeled with one of the isobaric tags from the set and the same peptide labeled with another of the isobaric tags) will elute together from the LC column. The isobaric tags also have the same mass and thus, the different versions of the labeled peptide will be indistinguishable in the MS1 spectra. However, upon fragmentation and separation in the MS2 spectra, each isobaric tag in the set releases a reporter ion having a different mass. By comparing the signal intensities of the reporter ions, relative quantification is obtained. Thus, the quantitative values are obtained from the MS2 spectra. Techniques for stable isotopic labeling and quantification from MS2 spectra include, for example, tandem mass tags (TMT) and isobaric tags for relative and absolute quantitation (iTRAQ). In a “six-plex” TMT experiment, six different samples may be compared simultaneously using a set of six isobaric tags. In an “eight-plex” iTRAQ experiment, eight different samples may be compared simultaneously using a set of eight isobaric tags.
In order to further increase the multiplexing capabilities of quantitative proteomics, i.e., the ability to analyze an even greater number of samples simultaneously, the stable isotopic labeling techniques involving quantification from MS1 spectra may be combined with techniques involving quantification from MS2 spectra. As one example, bio-duplicate samples could be distinguished in the MS1 spectra through the use of a light (e.g., unlabeled) and heavy isotopic label and each replicate could be analyzed at six time points with each time point distinguished in the MS2 spectra by a different six-plex isobaric tag, allowing the multiplex analysis of 12 samples at one time.
However, the potential of such multiplex experiments has been limited. As noted above, tandem mass spectrometer systems typically select only the most intense precursor ions for generation of the second mass spectrum. If a group of peaks (e.g., two peaks) as distinguished in the MS1 spectra and corresponding to an isotopically labeled peptide are not among the most intense peaks in the MS1 spectra, it is possible that not all peaks in the group will be selected for generation of MS2 spectra, precluding a quantitative comparison among the different samples. Moreover, even if all peaks in the group are selected, tandem mass spectrometer systems typically do not coordinate the timing of the MS2 spectra generated for the peaks. If significant time intervals separate the MS2 spectra generated for each peak in the group, the changing chemical background in the system may degrade the quantitative accuracy.